Cartilage, a highly specialized connective tissue, contains an extensive extracellular matrix and provides mechanical strength to resist compression in joints. In development, cartilage serves as the template for the growth and development of most bones. ECM molecules, such as perlecan, link protein, aggrecan, and type II collagen, are expressed during chondrocyte differentiation. Mutations of these genes and regulatory factors result in impaired cartilage formation and malformation of the limbs, craniofacial bones, and appendicular skeleton. Cartilage formation is initiated by mesenchymal cell condensation to form primordial cartilage followed by chondrocyte differentiation, which includes resting, proliferative, prehypertrophic, and hypertrophic chondrocytes. As a final step in endochondral bone formation, hypertrophic cartilage is invaded by blood vessels and osteoblasts, and the calcified cartilage is subsequently replaced by bone. Thus, spatial and temporal regulation of chondrocyte differentiation is essential in determining the length and width of skeletal components. We focus on protein factors that regulate chondrocyte differentiation and endochondral ossification.[unreadable] [unreadable] Although several factors have been identified as regulators for chondrocyte proliferation, it is still unclear how cell proliferation signals turn off and a commitment to differentiation is made. Pannexin 3 (Panx3), which we recently discovered, may be a candidate regulator for this process. We found that that Panx3 mRNA was expressed in the prehypertrophic zone where chondrocytes stop proliferation and are committed to differentiation into hypertrophic chondrocytes. Because of its unique expression pattern and potential gap junction function as a regulator for cell signaling, we hypothesized that Panx3 might control chondrocyte differentiation. We found that Panx3 expression was induced during chondrogenic ATDC5 cell differentiation into chondrocytes. Overexpression of Panx3 in ATDC5 cells promoted cell differentiation, whereas suppression of endogenous Panx3 by shRNA inhibited its chondrogenic differentiation in these cells. Panx3-overexpressing ATDC5 cells had increased release of intracellular ATP to the extracellular space via its hemichannel activity and reduced PTH-induced cell proliferation, intracellular cAMP levels, and phosphorylation of CREB, a downstream molecule of PKA that activates genes for proliferation. Our results suggest that Panx3 functions to switch chondrocyte cell fate from proliferation to differentiation by reducing the intracellular ATP/cAMP levels.[unreadable] [unreadable] TGF-beta regulates chondrocyte proliferation and differentiation. However, its in vivo function in cartilage development is not clear because of its diverse activities and redundant expression of multiple TGF-beta proteins. To define the precise role of TGF-beta signaling in cartilage development, we created conditional knockout (CKO) mice for ALK-5 (a type I receptor for TGF-beta). Chondrocyte-specific ALK-5 deficiency mediated by Col2a1-Cre caused severe defects in the axial cartilage in the spine and skull, while appendicular cartilage was relatively normal. The formation of intervertebral disks was defective, and segmentation of the upper vertebrae was disrupted. Although chondrocytes were differentiated, expression of extracellular matrix and glycosaminoglycan (GAG) levels were reduced. Perichondrium cells migrated into cartilage at the herniation site, suggesting reduced strength of mutant cartilage. We also created Dermo1-Cre-mediated ALK-5 CKO mice, in which Dermo-Cre is expressed in the progenitor mesenchyme. Mutant mice had similar axial skeletal abnormalities but more severely impaired appendicular skeletal elements. The mice developed multiple ectopic cartilaginous protuberances in growth plates and joint fusions. The perichondrium and periosteum were thin, and bone collar formation and endochondral ossification were reduced. These results suggest that TGF-beta signaling promotes chondrocyte proliferation and extracellular matrix synthesis and is required for the osteoblastic cell lineage.[unreadable] [unreadable] We found that the newly discovered dentin matrix molecule TM14 (fibulin-7) was expressed not only in teeth but also in cartilage. In situ hybridization revealed that TM14 mRNA was expressed in articular and proliferating chondrocytes, and in the perichondrium of growth plates. The TM14 protein was localized in the pericellular region of chondrocytes distinct from other cartilage matrix, such as type II collagen. We found that TM14 was induced during differentiation of chondrogenic ATDC5 cells. These results suggest that TM14 may play a role in chondrogenesis.